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J Biomed Biotechnol. 2004 December 1; 2004(5): 253–258. doi: 10.1155/S1110724304404136. | PMCID: PMC1082898 |
Sour Cherry (Prunus cerasus L) Anthocyanins
as Ingredients for Functional Foods Federica Blando,1* Carmela Gerardi,1 and Isabella Nicoletti2 1Istituto di Scienze delle Produzioni Alimentari, CNR, 73100 Lecce, Italy 2Istituto di Metodologie Chimiche, CNR, Area della Ricerca Roma 1, 00015 Montelibretti, Roma, Italy Received April 19, 2004; Revised June 10, 2004; Accepted June 15, 2004. In the recent years many studies on anthocyanins
have revealed their strong antioxidant activity and their possible
use as chemotherapeutics. The finding that sour cherries
( Prunus cerasus L) (also called tart cherries) contain
high levels of anthocyanins that possess strong antioxidant and
anti-inflammatory properties has attracted much
attention to this species. Here we report the preliminary results
of the induction of anthocyanin biosynthesis in sour cherry
callus cell cultures. The evaluation and characterization of the
in vitro produced pigments are compared to those of the
anthocyanins found in vivo in fruits of several sour cherry
cultivars. Interestingly, the anthocyanin profiles found in whole
fruit extracts were similar in all tested genotypes but were
different with respect to the callus extract. The evaluation of
antioxidant activity, performed by ORAC and TEAC assays, revealed
a relatively high antioxidant capacity for the fruit
extracts (from 1145 to 2592  μmol TE/100 g FW) and a
lower one for the callus extract (688 μmol TE/100 g FW). Anthocyanins, one of the major groups
of pigments belonging to the secondary metabolite group
of flavonoids, are often responsible for the orange, red, and
blue colors in fruits, vegetables, flowers, and other storage
tissues in plants. Thus, they have become important as food
additives. However, interest in anthocyanins has recently
intensified because of their possible health benefits. One of the
best known properties of flavonoids in general is their strong
antioxidant activity in metabolic reactions due to their ability
to scavenge oxygen radicals and other reactive species. This
feature makes flavonoids a potential tool for use in studies on
oxidative stress, the ageing process, and cancer [ 1],
especially since it has been reported that anthocyanins inhibit
the growth of cancer cells [ 2] and act as chemotherapeutics
for numerous diseases [ 3]. The finding that sour cherries
( Prunus cerasus L) contain significant levels of
anthocyanins [ 4] has attracted much attention to this
species. Anthocyanins from sour cherry have been shown to possess
strong antioxidant and anti-inflammatory activities [ 5] and
to inhibit tumor development in Apc Min mice and
the growth of human colon cancer cell lines [ 6]. Moreover,
cyanidin, the anthocyanin aglycon, has shown more efficient
anti-inflammatory activity than aspirin [ 5]. Although
cherry fruit tissue has recently been used in meat products for
improved nutritional qualities (less rancidity and the
inhibition of the formation of heterocyclic aromatic amines),
the use of purified anthocyanins extracted from cherry cells
cultured in vitro is an alternative to consider. The production
of anthocyanins extracted from cherry fruit is restricted to
their seasonal production; moreover, the fruit has too
high a value as a fresh fruit to be used for
anthocyanin extraction. Plant cells/tissue cultures offer an
opportunity for continuous production of plant metabolites.
Moreover, plant cell culture is an attractive production source,
since it is scalable according to specific needs [ 7] and
also gives greater potential for the manipulation of anthocyanin quality [ 8,
9]. Here we report the preliminary results of the induction of
anthocyanin biosynthesis in sour cherry ( P cerasus L)
callus cell cultures. The evaluation and characterization of
anthocyanin pigments produced in vitro as well as of those extracted
from the whole fruits of several cultivars are reported. The
anthocyanins pigment profiles in fruits and in callus cultures
producing anthocyanins were characterized by reverse-phase
high-performance liquid chromatography (RP-HPLC). This technique
coupled with a photodiode array detector has become the method of
choice for monitoring anthocyanin profiles [ 4,
10, 11].
Oxygen radical absorbance capacity (ORAC) assay and Trolox equivalent antioxidant capacity (TEAC) assay
on fruit and callus extracts have also been performed in order to evaluate the antioxidant activity of
the extracts. Chemicals All reagents (from Carlo Erba, Milan, Italy, if not otherwise
indicated) were of analytical reagent grade or HPLC grade, as
required. Anthocyanin standards were supplied by
Extrasynthese (Lyon, France). Plant material Fruits of sour cherry (P cerasus L) cv Amarena
Mattarello (AM), Visciola Ninno (VN), and
Visciola Sannicandro (VS) (genotypes from the local germplasm) were picked up in June 2003
on a local experimental field (Bari, Italy). Cherries were
flushed with nitrogen in freezer bags prior to storage at −20°C. Callus induction and anthocyanin production In vitro shoot culture of P cerasus L, cv AM,
was previously set up [ 12]. Callus cultures were induced from leaf segments on a callus
induction medium (CIM), containing Murashige and Skoog (MS)
mineral salts and vitamins, 30 g L −1 sucrose, 1 mg
L −1 α-naphthaleneacetic acid (NAA), and 0.1 mg
L −1 N 6-benzyladenine (BA). Leaf explants were taken from
plants grown in vitro on a plant multiplication medium (PMM)
[ 12]. The explants were incubated in a growth chamber at
25 ± 2 °C in the dark. Callus cultures were maintained
on the same CIM in the dark and transferred to a fresh CIM every three weeks. At the end of the growth cycle, callus cultures were transferred
to several types of media, later referred to as anthocyanin
induction media (AIM) [ 13], and then incubated under light
(Philips TLD/83,125 μmol m −2s −1),
with a 16 hour photoperiod. Anthocyanin producing calli were
harvested after two weeks of incubation under light, flushed with
nitrogen, and stored at −20 °C for further analysis. Extraction of the anthocyanins Pitted and frozen cherries (10 g) of each cv were ground twice
for 30 second in a Waring blender (Waring, Conn, USA) in the
presence of liquid N 2, thus providing a uniform powdered
sample. Frozen calli were pulverized with a pestle and mortar and
then treated as the homogenized fruits. A sample of the powder
(1 g) was centrifuged (Allegra 21 R centrifuge, Beckman Coulter,
Fullerton, Calif) at 10 000 g for 10 minutes at
4 °C. The supernatant juices were stored at −20 °C as stock solutions for the analysis of
antioxidant activity. Another sample of the same powder (3 g)
was extracted with a double volume of acidified methanol (0.01%
HCl) (v/v), at 4 °C, with stirring overnight. After extraction,
the colored liquid was separated from the solid
matrix and concentrated at 35 °C in vacuo and then dissolved
in the mobile phase used for HPLC analysis. HPLC/DAD analysis The profile of anthocyanins was determined using an HPLC system
consisting of a Model SCL-10AVP system controller, equipped with
a solvent delivery unit Model LC-10ADVP; an online vacuum
membrane degasser, Model DGU-14A; a column oven, Model
CTO-10ASVP; and a photodiode array detector
UV-Vis, Model SPD-10AVP, in conjunction with an LC workstation
Model Class VP 5.3 (all from Shimadzu, Milan, Italy).
Analytical separation of anthocyanin compounds was carried out
on a Polaris C18A column (150 × 2.0 mm, id
5 μm, Varian, Palo Alto, Calif) equipped with a
C18 guard column. The samples were introduced onto the column via
a Rheodyne Model 9125 nonmetal injection valve with a peak sample
of 5 μL volume. The temperature of the column oven was
set at 30 ± 0.1 °C. Solvent A was water : formic acid
(9 : 1 v/v); solvent B was acetonitrile : water : formic acid
(5 : 4 : 1 v/v). The percentage of solvent B was increased linearly
from 8% to 18% in 12 minutes, followed by elution under
isocratic conditions for the next 5 minutes, and by a second
linear gradient segment from 18% to 35% B in 13 minutes.
The column was reconditioned with the initial eluent for about
20 minutes. The solvent flow rate was 0.2 mL/min.
Acquisition range was set between 240 and 600 nm with a
sampling period of 0.32 second and a time constant of
0.64 second. The chromatogram was monitored at 518 nm.
The purity of the peaks was also monitored using the diode array
purity test system included in the software. Identification of anthocyanins The anthocyanin identification in sour cherry extracts was made
from matching UV-Vis spectra and retention times with
authentic standards. The quantities of different anthocyanins
were assessed from the peak areas and calculated as equivalents
of cyanidin 3-glucoside. The standard curve of this compound showed
excellent linearity over the concentration range of
4–50 mg/mL with correlation coefficient better than 0.9999
and nearly passed through the origin. Relative standard
deviations were less than 2%. Sour cherry samples were analyzed
in triplicate, and the mean peak areas of all anthocyanins were
used to determine the quantities present in the different cultivars and in the callus cell cultures. ORAC assay ORAC assays for fruit and calli juices were carried out following
the modified procedures of the method previously described by Ou et al
[ 14]. This
assay measures the ability of antioxidant components in test
materials to inhibit the decline in disodium fluorescein (FL)
(Sigma-Aldrich, St Louis, Mo) fluorescence that is induced by the
peroxyl radical generator, 2′,2′-Azobis (2-amidinopropane)
dihydrochloride (AAPH) (Wako Chemicals, Richmond, Va). The
reaction mixture contained in the final assay mixture
(0.7 mL total volume) FL (6.3 × 10 −8M) and
AAPH (1.28 × 10 −2M). All reagents were prepared
with 75 mM phosphate buffer, pH 7.4. The final volume was
used in a 10 mm wide fluorometer cuvette. FL, phosphate
buffer, and samples were preincubated at 37 °C for
15 minutes. The reaction was started by the addition of AAPH.
Fluorescence was measured and recorded every 1 minutes at the
emission length of 515 nm and excitation length
of 493 nm using a Shimadzu RF-5301PC (Columbia, Md)
until the fluorescence of the last reading declined to a value of less than or equal to
5% of the first reading. This usually took about
30 minutes. Phosphate buffer was used as the blank and 1, 5,
12.5 μM Trolox (Sigma-Aldrich, Steinheim, Germany) were used as the control
standards. Samples and Trolox calibration solutions were analyzed
in duplicate. The final ORAC values were calculated by using a
regression equation between the Trolox concentration and the net
area under the FL decay curve () and were
expressed as Trolox equivalents (TE) as micromole ( μmol) per
100 g of fresh weight (FW). ABTS radical cation decolorization assay This TEAC assay for fruit and callus juices was carried out
following the procedures previously described by Re et
al [ 15]. 2,2′-azino-bis
(3-ethylbenzothiazoline-6-sulfonic acid; (ABTS) (Sigma-Aldrich, St
Louis, Mo) was dissolved in water to a 7 mM concentration.
ABTS radical cation (ABTS •+) was produced by reacting
ABTS stock solution with 2.45 mM potassium persulfate (final
concentration) and allowing the mixture to stand in the dark at
room temperature for 12–16 hours before use. For the study of
fruit and callus juices, the ABTS •+ solution was
diluted with PBS, pH 7.4, to an absorbance of 0.70 (±
0.02) at 734 nm. Fruit and callus juices were diluted so
that, after the introduction of a 10 μL aliquot of each
extract into the assay, they produced between 20%–80%
inhibition of the blank absorbance. After addition of 1.0 mL
of diluted ABTS •+ solution to 10 μL of
extracts or Trolox standards (final concentration
0–15 μM) in PBS, the absorbance reading was taken up to
6 minutes. Appropriate PBS blanks were run in each assay. All
determinations were carried out twice. The final TEAC
decolorization assay values were calculated by using a regression
equation between the Trolox concentration and the percentage of
inhibition of absorbance at 734 nm after 6 minutes and
were expressed as TE as micromole ( μmol) per 100 g of FW. Sour cherry ( P cerasus L) is a temperate fruit with
marginal importance, even though since the 1980s this crop has
become more appreciated than the sweet cherry crop for reasons
such as minor agrobiological needs, the greater ease of
mechanical harvesting, and its numerous uses in the food industry.
Sour cherry may acquire new interest, mainly due to the fact that
it can be considered as a “functional food” because of its high
content of antioxidant compounds. Recent studies have revealed
that anthocyanins from sour cherry exhibit in vitro antioxidant
activities comparable to those from commercial products, such as
butylated hydroxyanisole (BHA) and butylated hydroxytoluene
(BHT), and superior to vitamin E at 2 mM concentration
[ 5]. In an anti-inflammatory assay, cyanidin (the aglycon of
the main tart cherry anthocyanins) showed better
anti-inflammatory activity than aspirin [ 5]. Thus, the
production of a “natural aspirin” could be a pharmaceutical
alternative for people with digestive tract ulcer or allergies to
aspirin and to nonsteroidal anti-inflammatory compounds. The interest in sour cherry anthocyanins has pushed us into a new
research project concerned with the in vitro production of
anthocyanin from the species and the characterization of the
secondary metabolites, compared to the in vivo products from the
fruits of different cultivars. Leaf explants cultured in the dark produced actively growing
callus cultures in a short time. The production of anthocyanins
was observed at different levels in most of the AIM tested. Even
in the CIM (control medium) anthocyanin production was
noticeable, confirming that an important factor for anthocyanin
induction is light. Takeda [ 16] reported that, in carrot cell
cultures, light irradiation induced the expression of enzymes of
the phenylpropanoid metabolic pathway, such as phenylalanine
ammonia-lyase and chalcone synthase, at the transcription level. During several subcultures (nearly ten), a stepwise selection of callus cultures allowed
the isolation of a cell line with high and homogeneous anthocyanin production
(). The pigmented callus cultures as well as the fruit extracts from different local cultivars
were used for the evaluation and quantification of the anthocyanins. shows the anthocyanin profiles found in
AM cherry extract and in callus extract. The spectra of the separated anthocyanins in
sour cherry samples were very similar to those of cyanidin
3-glucoside, the standard used for their quantification
(). Cyanidin 3-glucosylrutinoside, cyanidin
3-sophoroside, cyanidin 3-rutinoside, and cyanidin 3-glucoside
were identified as major components in the analyzed samples in
agreement with the findings previously reported in the literature
[ 4]. The chromatographic results showed that, in the
analyzed sour cherry, the same compounds were present in all the
cultivars but at different levels. The total anthocyanin content
ranged from 27.8 to 80.4 mg/100 g ( Table 1). | Table 1Total anthocyanin content and composition of sour cherry
(P cerasus L) fruit extracts of Visciola Ninno, Amarena
Mattarello, and Visciola Sannicandro cultivars and of callus
culture extract. Anthocyanin composition was determined as a
percentage of total (more ...) |
As reported in Table 1, the total anthocyanin content
was much higher in the fruit extracts than in the callus extract.
Our in vitro system is at a preliminary stage of development and
it is important to set up the best possible microenvironmental
conditions to improve the anthocyanin production. The callus
cultures we selected are capable of producing 20-fold less
anthocyanin than the fruit of the same field-grown cultivar
(AM) ( Table 1). Our aim is now to
greatly improve the pigment production, to make the in vitro
process economically viable and an alternative to the field-grown
material. To raise the efficiency it is important, for example, to
induce anthocyanin production not only at the surface but even inside the callus. Although total anthocyanin content in our cultivars varied,
depending on the genotype, each cultivar profile contained the
same compounds in quite similar proportions. Cyanidin
3-glucosylrutinoside was the main anthocyanin found, followed by
cyanidin 3-rutinoside, cyanidin 3-sophoroside, and cyanidin
3-glucoside. The anthocyanin profile found in fruit extracts was
similar in all tested genotypes and was quite different from that
found in the callus extract. In the callus extract only cyanidin
3-glucoside and cyanidin 3-rutinoside were present, the first one
at a very high proportion (72.14%) ( Table 1). This
aspect reveals the ability of the in vitro cell to modulate the
anthocyanin metabolism towards less evolved molecular structures.
It is reported that the metabolic flux of in vitro systems is
often simplified but could be driven towards the accumulation of
specific compounds with interesting characteristics [ 9,
17],
thus providing a powerful tool for biotechnological applications. Since it has been shown that the role of fruit and vegetables in
protection against cancer, cardiovascular disease, and cerebrovascular disease is
to be attributed to the various antioxidant compounds contained in these
foods, and that the biological activities of anthocyanins could also be due to their
antioxidant properties, the evaluation of the antioxidant
capacity of anthocyanin containing fruits or anthocyanin
extracts is an important parameter for the suitable formulation of functional foods. Several methods have been developed to measure the total
antioxidant capacity of various biological samples. Among them,
ORAC and TEAC are the most important. We used an improved ORAC assay using FL as a fluorescent
probe, since it has been shown that the previously used probe, the
β-Phycoerythrin, gave problems in terms of photostability
and reproducibility [ 18]. The total antioxidant capacity of fruit extracts, measured as
ORAC FL, ranged from a low 1145 to 1916 μmol
TE/100 g of FW (). These values are not
comparable with those reported in several papers in which
β-Phycoerythrin is used as the fluorescent probe, as
described by Ou et al [ 14]. In TEAC assay, values
showed a range of 2000–2600 μmol TE/100 g of FW
(). Few reports deal with the evaluation of
the antioxidant activity of fruit extracts using the TEAC
procedure [ 19]. The values found in sour cherry fruits are
comparable to those found in some berry fruits, for example,
strawberry, and are higher than in apple and kiwiafruit
[ 20]. The total antioxidant capacity of the callus extracts
was lower than that of fruits, in both ORAC and TEAC assays
(630–746 μmol TE/100 g of FW)
(Figures and ).
However, if we compare the total antioxidant
capacity of the callus to that of the fruit, the value is nearly
4-fold less, whereas it was 20-fold less in terms of anthocyanin
content. The antioxidant capacity can be supported even by other
polyphenolic compounds contained in sour cherry fruits, as
reported by Wang et al, [ 21]. These compounds could be at
a high level in callus cultures, thus contributing to the total antioxidant capacity. The values found for sour cherry in the ORAC and TEAC assays were
quite similar, even though the two methods are based on different
reaction mechanisms. TEAC is based on the inhibition of the
absorbance of the radical cation of ABTS by antioxidants. The
antioxidants are oxidized by the oxidant ABTS•+, with
a single electron transfer (SET) reaction from the antioxidant
molecule to the oxidant. The ORAC assay is based on a hydrogen
atom transfer (HAT) reaction, with a peroxyl radical
ROO• that abstracts a hydrogen atom from the
antioxidants, thus retarding or inhibiting the reaction between
the ROO• and the target molecule probe. The different
reaction mechanisms of the two assays could explain the different
rank order of the three cultivars. The ORAC value for cv VN was
different from the TEAC value, whereas the values for the other
materials were comparable. The antioxidant activity assays used juice extracts while the
anthocyanin analysis used methanolic extracts in order to perform
both assays on samples extracted with the most suitable solvent for each method.
Therefore, it is possible to compare the anthocyanin content to
the values found in the antioxidant capacity assays. The trend
shown by the ORAC assay fitted that of the anthocyanin content
particularly for fruits. The higher anthocyanin contents were for
cv AM and cv VS, which showed the higher ORAC values. For callus,
we discussed above the possible contribution of antioxidant
compounds other than anthocyanins. Moreover, the antioxidant
melatonin was recently identified in two cultivars (Balaton and
Montmorency) of tart cherry [ 22]. This finding
provides an interesting link with the
anecdotal consumption of cherries as a health promoting food, and represents a further
reason for studying this fruit species. 1. Rice-Evans C. Screening of phenolics and flavonoids for antioxidant activity. In: Packer L, Hiramatsu M, Yoshikawa T, editors. Antioxidant Food Supplements in Human Health. San Diego, Calif: Academic Press; 1999. pp. 239–253. 2. Kamei H, Kojima S, Hasegawa M, Umeda T, Terabe K, Yukawa T. Suppressive effect of flavonoid extracts from flower petals on
cultured human malignant cells. J Clin Exp Med. 1993;164:829–830. 3. Bomser J, Madhavi D.L, Singletary K, Smith M.A. In vitro anticancer activity of fruit extracts from Vaccinium species. Planta Med. 1996;62(3):212–216. [PubMed] 4. Wang H, Nair M.G, Iezzoni A, Strasburg G.M, Booren A.M, Gray J.I. Quantification and characterization of anthocyanins in Balaton tart cherries. J Agric Food Chem. 1997;45(7):2556–2560. 5. Wang H, Nair M.G, Strasburg G.M, et al. Antioxidant and antiinflammatory activities of
anthocyanins and their aglycon, cyanidin, from tart cherries. J Nat Prod. 1999;62(2):294–296. [PubMed] 6. Kang S.Y, Seeram N.P, Nair M.G, Bourquin L.D. Tart cherry anthocyanins inhibit tumor development in ApcMin mice and reduce proliferation of human colon cancer cells. Cancer Lett. 2003;194(1):13–19. [PubMed] 7. Smith M.A, Pépin M. Stimulation of bioactive flavonoid production in
suspension and bioreactor-based cell cultures. In: Altman A, Ziv M, Izhar S, editors. Plant Biotechnology and In Vitro Biology in the 21st Century. Dordrecht: Kluwer Academic Publishers; 1999. pp. 333–336. 8. Curtin C, Zhang W, Franco C. Manipulating anthocyanin composition in Vitis vinifera suspension
cultures by elicitation with jasmonic acid and light irradiation. Biotechnol Lett. 2003;25(14):1131–1135. [PubMed] 9. Plata N, Konczak-Islam I, Jayram S, McClelland K, Woolford T, Franks P. Effect of methyl jasmonate and p-coumaric acid on anthocyanin
composition in a sweet potato cell suspension culture. Biochemical Engineering Journal. 2003;14(3):171–177. 10. Hong V, Wrolstad R.E. Characterization of anthocyanin-containing
colorants and fruit juices by HPLC/photodiode array detection. J Agric Food Chem. 1990;38(3):698–708. 11. Goiffon J-P, Mouly P.P, Gaydou E.M. Anthocyanic pigment determination in red fruit juices,
concentrated juices and syrups using liquid chromatography. Anal Chim Acta. 1999;382(1-2):39–50. 12. Blando F. In vitro propagation of Prunus cerasus L. Italus Hortus. 2002;9(3):16–17. 13. Blando F, Gala R, Gerardi C, Druart P, editors. Sour cherry (Prunus cerasus L) production towards the utilization
for a new century. In: Proceedings of the 26th International Horticultural Congress; 2002; Toronto. IHS ed; p. 180. 14. Ou B, Hampsch-Woodill M, Prior R.L. Development and validation of an improved oxygen radical
absorbance capacity assay using fluorescein as the fluorescent probe. J Agric Food Chem. 2001;49(10):4619–4626. [PubMed] 15. Re R, Pellegrini N, Proteggente A, Pannala A, Yang M, Rice-Evans C. Antioxidant activity applying an improved ABTS radical cation
decolorization assay. Free Radic Biol Med. 1999;26(9-10):1231–1237. [PubMed] 16. Takeda J. Light-induced synthesis of anthocyanin in carrot cells
in suspension. J Exp Bot. 1991;41:749–755. 17. Konczak-Islam I, Nakatani M, Yoshinaga M, Yamakawa O. Effect of ammonium ion and temperature on anthocyanin composition
in sweet potato cell suspension culture. Plant Biotechnol. 2001;18(2):109–117. 18. Huang D, Ou B, Hampsch-Woodill M, Flanagan J.A, Prior R.L. High-throughput assay of oxygen radical absorbance capacity (ORAC)
using a multichannel liquid handling system coupled with a microplate
fluorescence reader in 96-well format. J Agric Food Chem. 2002;50(16):4437–4444. [PubMed] 19. Proteggente A.R, Pannala A.S, Paganga G, et al. The antioxidant activity of regularly consumed fruit and
vegetables reflects their phenolic and vitamin C composition. Free Radic Res. 2002;36(2):217–233. [PubMed] 20. Lister C.E, Wilson P.E, Sutton K.H, Morrison S.C. Understanding the health benefits of blackcurrants. Acta Hort. 2002;585:443–449. 21. Wang H, Nair M.G, Strasburg G.M, Booren A.M, Gray J.I. Novel antioxidant compounds from tart cherries (Prunus cerasus). J Nat Prod. 1999;62(1):86–88. [PubMed] 22. Burkhardt S, Tan D.X, Manchester L.C, Hardeland R, Reiter R.J. Detection and quantification of the antioxidant melatonin in
Montmorency and Balaton tart cherries (Prunus cerasus). J Agric Food Chem. 2001;49(10):4898–4902. [PubMed]
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